Abstract
Lentiviral vectors have rapidly become a favorite tool for research and clinical gene transfer applications which seek to permanently introduce alterations in the genome. This status can be attributed primarily to their ability to transduce dividing as well as quiescent cells. When coupled with internal promotor selection to drive expression in one cell type but not another, the ease with which the vectors can be pseudotyped to either restrict or expand tropism offers unique opportunities previously unavailable to the researcher to manipulate the genome. Although LV can be produced from stable packaging cell lines and/or in suspension culture, by and far, most LV vectors are produced using adherent 293 T cells grown in plasticware and production plasmids transiently transfected with either PEI or Calcium Phosphate. The media is usually changed and un-concentrated vector supernatant collected between 24 and 48 h post-transfection. The supernatant may then be purified by Mustang Q chromatography, concentrated by Tangential Flow Filtration, and finally diafiltered into the final formulation buffer of choice. Here we describe a pilot scale method for the manufacture of a Lentiviral vector that purifies and concentrates approximately 6 L of un-concentrated LV supernatant to approximately 150 mL. Typical titers for most vector constructs range between 1 × 108 and 1 × 109 infectious particles per mL. This method may be performed reiteratively to increase total volume or can be further scaled up to increase yield.
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Acknowledgments
Our facility is generously supported by the Cincinnati Children’s Hospital Research Foundation. Most importantly, we would like to acknowledge the hard work and dedication of the entire staff of the Translational Core Laboratories at Cincinnati Children’s Hospital; without it, our contributions to the gene therapy field would not be possible.
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Tinch, S., Szczur, K., Swaney, W., Reeves, L., Witting, S.R. (2019). A Scalable Lentiviral Vector Production and Purification Method Using Mustang Q Chromatography and Tangential Flow Filtration. In: Manfredsson, F., Benskey, M. (eds) Viral Vectors for Gene Therapy. Methods in Molecular Biology, vol 1937. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9065-8_8
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DOI: https://doi.org/10.1007/978-1-4939-9065-8_8
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